ABSTRACT Our work to date on the current grant has yielded the following results: a) single mouse parotid cells were found to form three-dimensional (3D) cell clusters displaying TJ and agonist-induced secretory responses when grown on Growth Factor-Reduced Matrigel (GFR-MG) in combination with Fibrin Hydrogel1 (FH), b) the ratio of GRF-MG and FH was optimized with the ultimate goal of creating a functional and clinically safe scaffold1, c) our salivary cell isolation method was improved to maintain secretory granules, form TJ and facilitate cell survival, d) mouse submandibular gland (mSMG) cells, rather than parotid or sublingual glands, were found to be the best choice for creating salivary cell clusters displaying organized morphology, e) critical components were identified within GFR-MG (i.e., EGF and IGF-1) that enhance salivary gland (SG) differentiation when polymerized to FH, f) EGF and IGF-1 were found to be incapable of independently producing organized cell clusters; however, this goal was achieved using Laminin-1 (L1), g) peptides (corresponding to four L1 regions that promote intact SG formation) were synthesized and conjugated to FH, h) specific L1 peptides induced formation of acinar spheres in the rat parotid cell line Par-C10 (as compared to cells grown on FH alone), i) addition of a conditioned medium from human hair follicle mesenchymal stem cells (hHF-MSC CM) improved salivary cell cluster organization and lumen formation in mSMG cells, j) salivary cell clusters maintained acinar, ductal and myoepithelial cells while responding to secretory agonists, k) lumen formation was impeded with the blocking of FGF10 in hHF-MSC CM, l) high amounts of FGF2 were detected in a hHF-MSC CM in which intact salivary cell clusters were grown, m) L1 peptides chemically conjugated to fluorescent FH were applied to wounded mSMG in vivo to form new salivary tissue, as compared to FH alone or no scaffold, and mice were healthy after the treatment. Taken as a whole, the above studies indicate we have accomplished the majority of our initial goals during the first three years of this grant and found viable alternatives for the difficulties encountered (e.g., use of L1 peptides to enhance FH and improve salivary cell clusters formation, as noted above). In the coming grant period, we will refine our design for a clinically safe environment as follows: a) produce a FH modified with L1 peptides and growth factors, b) grow differentiated salivary cell clusters in these matrices and c) use modified FH scaffold to form new and functional tissue in vivo (with aim of later applying these findings to humans with SG dysfunction due to Sjgren's syndrome as well as head and neck ?-irradiation treatments).